Creatinine biosensor

ABSTRACT

The invention relates to a method for producing biosensors that comprise at least two enzymes for the amperometric determination of enzymatically cleavable substances, such as creatinine, in biological liquids, the enzymes being immobilized on a working electrode. An enzyme is applied on the working electrode with one or more surface-active substances in an aqueous solution and is allowed to dry. The at least second enzyme is chemically immobilized thereon in a subsequent step, thereby allowing for shorter response times and stronger signals of the biosensor.

[0001] The invention relates to a method for producing biosensorscomprising at least two enzymes, for the amperometric determination ofenzymatically degradable substances in biological liquids, the enzymesbeing immobilized on a working electrode. Furthermore, the inventionrelates to a biosensor, in particular for the determination ofcreatinine.

[0002] The determination of enzymatically degradable substances, such ascreatinine, glucose etc., by means of sensors in biological liquids, forexample in blood, urine, plasma, serum and liquor, is preferably carriedout via biosensors comprising immobilized enzymes. From the literature,several electrochemical and photometric methods of determining thosesubstances are known.

[0003] In that way, creatinine may be potentiometrically determined, forexample by means of the enzyme creatinine deiminase, involving asubsequent determination of the ammonium content. Another methodconsists in determining the creatinine concentration by means of anenzyme cascade using the enzymes creatininase, creatinase and sarcosineoxidase, with hydrogen peroxide (H₂O₂) being finally measured at anamperometric electrode.

[0004] The invention relates to a method for producing biosensors thatfunction in accordance with the last-mentioned principle. In doing so,the enzymes must be coimmobilized in order to allow for the conversionof creatinine to the amperometrically detectable molecule hydrogenperoxide. The conversion of creatinine to hydrogen peroxide is carriedout according to the following reaction steps:

creatinine+H₂O−creatininase→creatine

creatine+H₂O−creatinase→sarcosine+urea

sarcosine+O₂+H₂O−sarcosine oxidase→glycin+formaldehyde+H₂O₂

[0005] At the amperometric electrode, hydrogen peroxide is oxidizedanodically against Ag/AgCl at −350 mV. The current flowing thereby isproportional to the creatinine concentration.

H₂O₂−350 mV→2 protones+2 electrones+O₂

[0006] The oxygen recovered during the electrode reaction is then usedfor the oxidation of the sarcosine.

[0007] In the state of the art, various ways of immobilizing the threeused enzymes are known. According to T. Tsuchida, K. Yoda, Clin. Chem.29/1, p.51, 1983, all three enzymes are cross-linked withglutardialdehyde.

[0008] However, that method of immobilization entails the drawback that,by means of a sensor produced in such a manner, only a low signal heightis reached, i.e. only a slight change in current is ascertainable sincethe sarcosine oxidase immobilized in that manner loses almost its entireactivity. However, a greatest possible signal height is particularlyimportant especially for the determination of creatinine, since theconcentration of creatinine is very small, especially in blood(approximately 50 μM), and, in addition, creatinase is available onlywith very small specific activities (20 iu/mg at the most). Furthermore,a sensor immobilized in such a manner exhibits long response times.

[0009] In U.S. Pat. No. 5,466,575, a method is described in whichsarcosine oxidase and creatininase are immobilized in a fish gelatincapable of being subjected to light-induced crosslinking andsubsequently are superimposed by creatinase in a film-formingpolyvinyl-acetate-co-vinyl-alcohol latex.

[0010] In this connection, however, the complicated light-inducedcrosslinking which prevents a simple production of the biosensorconstitutes a drawback.

[0011] The object of the invention is to provide a method of theinitially mentioned kind which overcomes the above-mentioned drawbacksand difficulties. In particular, the method according to the inventionis to allow for a simple production of a biosensor with which both shortresponse times and great signal heights are achievable. In particular,immobilizing the enzymes at room temperature is to be possible.

[0012] According to the invention, that object is achieved in that anenzyme together with one or more surface-active substances in an aqueoussolution is applied on the working electrode and is allowed to dry, andthe at least second enzyme is chemically immobilized thereupon in asubsequent step.

[0013] For the purposes of the present description and patent claims,the term “surface-active substances” is to cover substances whichpossess surface-active characteristics, such as detergents and alcohols,for example glycerine.

[0014] Preferably, polyalcohols and/or detergents, preferably non-ionictensides, are used as surface-active substances.

[0015] It has been found out that, by means of those additives, themeasured current is increased by a factor of about 40 in comparison tobiosensors comprising three equally immobilized enzymes.

[0016] Suitably, the at least second enzyme is immobilized by means ofcrosslinking, covalent binding or matrix inclusion. Preferably,immobilization is brought about by glutardialdehyde.

[0017] In a preferred embodiment, a cover membrane is applied afterimmobilization of the enzymes. Such a membrane consisting, for example,of nafion, PVC copolymer or cellulose acetate advantageously increasesthe linearity of the sensors and, in addition, causes a reduction ofinterfering influences.

[0018] A biosensor according to the invention which comprises a working,a reference and a counter electrode and whose enzymes have beenimmobilized by means of the method according to the invention ischaracterized in that the reference electrode is an Ag/AgCl electrode,the counter electrode is a carbon electrode, the working electrodeconsists of carbon, metal, metal oxides or a mixture of carbon and metalor metal oxides and the electrodes are applied on a nonconductingsubstrate.

[0019] In particular, a biosensor according to the invention for thedetermination of creatinine is characterized in that sarcosine oxidaseis adsorbed on the working electrode and creatininase and creatinase areimmobilized thereupon.

[0020] In a preferred embodiment, the biosensor is made up of twothree-electrodes systems, the first electrode system comprising theenzymes creatininase, creatinase and sarcosine oxidase and serving forthe determination of the sum of creatinine and creatine and the secondelectrode system comprising the enzymes creatinase and sarcosine oxidaseand serving for the determination of creatine, whereby the result of thesecond electrode system is deducted from the result of the first one forthe determination of creatinine.

[0021] Advantageously, the biosensor comprises a further electrodesystem serving for the elimination of electrochemical interferences.

[0022] In the following, the invention is explained further by the aidof the following examples:

EXAMPLE 1

[0023] Example 1 shows the improvement of the signal height byincreasing the activity of the sarcosine oxidase when adding, inaccordance with the invention, surface-active substances, as opposed tothe prior art.

[0024] Sarcosine oxidase dissolved in water (prior art) as well as inwater with water-soluble, surface-active components (in the instantcase, glycerine as well as three non-ionic tensides) being added wasdropped onto the amperometric base sensor and was allowed to dry at roomtemperature. Upon polarization of the electrode, the current wasmeasured on 1 mM sarcosine. The measuring result is shown in Table 1.TABLE 1 Current on 1 mM Sarcosine oxidase (SOx) Additive sarcosine 54.2mg SOx in 0.5 ml H₂O none 2 nA 54.2 mg SOx in 0.5 ml H₂O 5.0% glycerine80 nA 54.2 mg SOx in 0.5 ml H₂O 0.5% Tween 20 70 nA 54.2 mg SOx in 0.5ml H₂O 0.5% Triton X100 90 nA 54.2 mg SOx in 0.5 ml H₂O 0.5% Brij 35 85nA

[0025] It was found that the current on 1 mM sarcosine could beincreased by a factor of about 40 by means of detergents or glycerine,respectively.

[0026] Apparently, that enormous increase in current has been achievedsince, during drying, the enzyme is protected in the best possible wayby the additives and since the surface-active characteristics of theadditives result in a better and closer bonding with the porous textureof the hydrogen peroxide electrode.

EXAMPLE 2

[0027] Example 2 shows the improvement of the signal height as well asthe shortening of the response time of a biosensor according to theinvention in comparison to a biosensor produced in accordance with theprior art (T. Tsuchida).

[0028] Two complete creatinine sensors were produced, whereby, in caseof the first sensor, all three enzymes together were cross-linked withglutardialdehyde and, in case of the second sensor, sarcosine oxidasewith Tween 20 was first applied on the base electrode and subsequentlycreatininase and creatinase were immobilized thereupon withglutardialdehyde. The resulting currents and response times,respectively, for measuring creatinine as well as sarcosine are listedin Table 2. TABLE 2 Current on 1 mM Current on 1 mM Response Sarcosineoxidase creatinine sarcosine time (T90) in glutardialdehyde 120 nA  140nA 80 s (sensor 1) in Tween 20 420 nA >500 nA 10 s (sensor 2)

[0029] By means of the immobilization according to the invention of theenzymes, a by far stronger current was measured. The response time ofthe sensor produced according to the invention was also clearly shorterthan that of the sensor known in the prior art.

EXAMPLE 3

[0030] In example 3, the production of a creatinine biosensor accordingto the invention is described.

[0031] By means of the serigraphy process, Ag-strip conductors forreference, counter and working electrodes were printed on anelectrically nonconducting substrate made of a synthetic or ceramicmaterial. The reference electrode is produced from an Ag/AgCl-paste atleast in the sensor area. A layer of carbon paste is printed on thecounter electrode within its measuring area. By means of the same carbonpaste, the Ag-strip conductor of the working electrode is extended intothe measuring area. In the measuring area of the working electrode, amixture of 5% manganese dioxide in carbon paste is printed as a workingelectrode. Subsequently, the entire system with the exception of theelectrode spots lateron to be contacted with a liquid and the stripconductors serving for the signal tap is repeatedly coated with aninsulating varnish. Following that, sarcosine oxidase in a Tween 20solution is dropped onto the working electrode and is allowed to dry.Thereupon, the other enzymes are immobilized by means ofglutardialdehyde. In order to increase linearity and to reduceinterfering influences, a cover membrane is applied.

[0032] In order to be able to determine creatinine without anyinterferences, at least two three-electrodes systems are necessary; onesystem comprising the enzymes creatininase, creatinase and sarcosineoxidase which determines creatinine and creatine as well another onecomprising the enzymes creatinase and sarcosine oxidase which serves forthe determination of creatine. Since creatine acts as an interferingsubstance in blood, the measured creatine value has to be deducted fromthe measured value of the creatinine electrode, which is composed ofcreatine and creatinine.

[0033] In order to eliminate further electrochemical interferences, athird electrode system may be used solely with immobilized sarcosineoxidase (creatine is replaced by an inactive protein, for examplealbumin).

1. A method for producing biosensors comprising at least two enzymes,for the amperometric determination of enzymatically degradablesubstances, such as creatinine, in biological liquids, the enzymes beingimmobilized on a working electrode, characterized in that an enzymetogether with one or more surface-active substances in an aqueoussolution is applied on the working electrode and is allowed to dry, andthe at least second enzyme is chemically immobilized thereupon in asubsequent step.
 2. A method according to claim 1, characterized in thatpolyalcohols and/or detergents, preferably non-ionic tensides, are usedas surface-active substances.
 3. A method according to claim 1 or 2,characterized in that the at least second enzyme is immobilized by meansof crosslinking, covalent binding or matrix inclusion.
 4. A methodaccording to claim 3, characterized in that the at least second enzymeis immobilized by means of glutardialdehyde.
 5. A method according toany of claims 1 to 4, characterized in that a cover membrane is appliedafter immobilization.
 6. A biosensor comprising a working, a referenceand a counter electrode, produced by means of the method according toany of claims 1 to 5, characterized in that the reference electrode isan Ag/AgCl electrode and the counter electrode is a carbon electrode andthe working electrode consists of carbon, metal, metal oxides or amixture of carbon and metal or metal oxides, the electrodes beingapplied on a nonconducting substrate.
 7. A biosensor according to claim6 for the determination of creatinine, characterized in that sarcosineoxidase is adsorbed on the working electrode and creatininase andcreatinase are immobilized thereupon.
 8. A biosensor according to claim7, characterized in that it is made up of two three-electrodes systems,the first electrode system comprising the enzymes creatininase,creatinase and sarcosine oxidase and serving for the determination ofthe sum of creatinine and creatine and the second electrode systemcomprising the enzymes creatinase and sarcosine oxidase and serving forthe determination of creatine, whereby the two results are subtractedfor the determination of creatinine.
 9. A biosensor according to claim 8which comprises a further electrode system serving for the eliminationof electrochemical interferences.